Rabies virus vaccine

ABSTRACT

A novel rabies virus strain No. 675 deposited in the Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under number CNCTO AO 4/77, novel living or inactivated cell culture vaccines derived therefrom, a process for the isolation of the virus strain as well as a process for the preparation of the said vaccines and novel method of immunizing warm-blooded animals against rabies.

STATE OF THE ART

It is known to combat rabies viral infections in warm-blooded animals bypreventive parenteral administration of vaccines such as LEP (Low EggPassage) Flury or HEP (High Egg Passage) Flury vaccines which areobtained as described below.

On Jan. 29, 1939 a girl name Flury died in the State of Georgia withrabies being the diagnosed cause of death after infection by a rabiddog. The rabies virus was isolated from the brains, lachrymal gland andthe salivary gland of the girl by means of intracerebral inoculationinto white mice as described in Leach et al., [Amer. J. Trop. Med.(1940), Vol. 20 p. 335].

The brain material from the mice was inoculated intracerebrally into oneday old chickens and 136 intracerebral passages in chickens wereperformed subsequently, according to Koprowski et al. [J. Immunol.(1948), Vol. 60 p. 533]. After two additional intracerebral passages inchickens, the rabies virus was adapted to chicken embryos by a yolk-sacinoculation. After 60 yolk-sac passages, the virus appeared to bepractically non-pathogenic for a number of mammals and is available atthis serial passage level as rabies vaccine for dogs under the name ofLEP Flury vaccine. After additional serial passages in chicken embryos(up to 170-174 passages), the virus appeared to have lost much morepathogenicity so that two week old laboratory mice were not killed afterintracerebral administration although suckling mice were still killed.This vaccine is available under the name of HEP Flury vaccine (High EggPassage Flury vaccine).

Although these LEP and HEP vaccines have been effective in preventingrabies infection when used as pre-exposure vaccines, the need for otherrabies vaccines does exist as demonstrated by recent new controlmeasures at the national and international level. For instance, thecontrol of sylvatic rabies in developed countries where this disease hasbeen endemic for a long time or where it has been introduced morerecently is one of the difficult and ecologically complicated existingsituations for which no effective and economical control method isavailable.

Also the post-exposure vaccine treatment of human beings has creatednumerous problems in rabies-infected countries throughout the world. Forthese reasons, research in rabies have been directed to the problems ofpost- and pre-exposure vaccination and immunization by a non-parenteral,preferably oral, route.

OBJECTS OF THE INVENTION

It is an object of the invention to provide a novel rabies strain and anovel process for the preparation thereof.

It is a further object of the invention to provide a novel rabiesvaccine living or inactivated and processes for obtaining the same.

It is an additional object of the invention to provide novel methods ofimmunizing warm-blooded animals against rabies.

These and other objects and advantages of the invention will becomeobvious from the following detailed description.

THE INVENTION

The novel rabies virus strain of the invention is No. 675 deposited inthe Czechoslovak National Collection of Type Culture of the Institute ofHygiene and Epidemiology in Prague under number CNCTC AO 4/77 and is themost apathogenic existing rabies vaccine strain so far known. It offersthe possibility of protection after exposure to street virus infectioneven using one single adequate dose of vaccine. The inactivated vaccineprepared from this strain 675 also protects after exposure, but to alower degree. The living vaccine possesses a very high degree ofantigenicity which is almost equalled by the inactivated type ofvaccine. The living and inactivated vaccines are cell culture-typevaccines almost completely free from foreign proteins, which diminishessubstantially, if not completely, the degree of undesirable sidereactions.

The new virus strain was isolated as follows: The virus of the HEP Fluryvaccine was propagated in primary or secondary SPF chicken embryofibroblasts and was also identified as rabies virus with the aid ofspecific immunofluorscence of these infected fibroblasts, by a mouseprotection test through intraperitoneal vaccination followed by theadministration of a standardized rabies challenge virus (CVS) and by invitro seroneutralization tests with known positive serum (InternationalRabies Reference Serum).

It will be appreciated that for the propagation of the starting HEPFlury vaccine also other rabies susceptible cell lines and diploid cellstrains may be applied instead of the SPF chicken embryo fibroblasts.

By the use of the plaque assay method in chicken embryo fibroblasts(SPF) according to Yoshina et al., [Arch. ges. Virusforsch., (1966),Vol. 18 p. 370]and the plaquing cell suspension method according toBijlenga et al., [Bull. Soc. Sci. Vet. et Med. Comparee, Lyon, (1974),Vol. 76, p. 429], it was possible to isolate a plaque which clearlydiffered from the starting virus population in its cytopathic effect onthe infected cells. This plaque was cloned three times in order toobtain a homogeneous and pure virus population.

The originally isolated plaque was allocated the number 675 and thestrain after purification as rabies vaccine strain No. 675. A sample ofthis strain was deposited with the Czechoslovak National Collection ofType Cultures of the Institute of Hygiene and Epidemiology in Prague onJan. 13, 1977, and is recorded there under CNCTC Number AO 4/77.

The strain can be distinguished from the original HEP Flury virus inrespect of the following characteristics: (a) A very pronouncedcytopathic effect on in vitro cell cultures, an effect which is notcaused by the HEP Flury virus: (b) Shortened primary virus cycle,varying between 9 and 11 hours, causing a more rapid appearance ofplaques; (c) Very clear plaques, which are on the average somewhatlarger (1 mm larger) in diameter than the plaques of the HEP virus; (d)Enhanced attaching capacity to the surface of cells in vitro, whichcharacteristic is essential for an adequate oral vaccine; (e) Fasterproduction of interferon in vivo after vaccination because of theproperties as mentioned hereinbefore. This property is important forpost-exposure vaccination; (f) Higher titers are obtained in cellcultures; (g) By clone purification, homogeneous virus populations areobtained resulting in a decreased risk of possible reversion of thevirus strain to its origial virulence, which reversion may bepractically excluded. (h) It is the most apathogenic rabies strain sofar available.

It will be appreciated that these characteristics as indicated under(a-h) show a clear cut distinction from the original HEP Flury virus. Itwill also be appreciated that the present invention includes within itsscope artificial mutant strains derived from virus strain No. 675 andhaving the properties of strain No. 675 described above. Due to theabove mentioned characteristics, particularly (g), the preparation ofvaccine batches having a constant quality is facilitated, i.e. vaccinesof improved consistency can be prepared according to the invention fromthe virus strain No. 675.

The vaccines which form another feature of the present invention showthe following advantages: (1) As with the original HEP Flury vaccine,the live virus vaccine of the invention does not multiply afteradministration and no residual virus can be detected; (2) The vaccinescaused very rapid interferon production and other additional, unknowninterfering properties, e.g. cellular mediated immunity. A singleadministration of vaccine after experimental infection with a naturallyoccuring virus can provide full protection.

In view of the very rapid production of interferon and cellular mediatedimmunity, the application of the vaccines of the invention for theprotection of recently exposed animals during outbreaks of rabies is nowfeasible. Particularly, the possibility of post-exposure vaccinetreatment of humans has been improved considerably as the vaccines havebeen shown to be highly effective in animals after exposure to infectionand can even cure animals in a very advanced state of infection. (3)Non-parenteral administration e.g. by the oral route of the livevaccine, facilitates its use in organized vaccination compaigns.

The living type of this vaccine is very suitable for oraladministration. Oral administration is desirable for the immunization(to be called hereinafter "oral vaccination") of eg. foxes. Oral andother non-parenteral routes of administration such as intestinal orintranasal administration by spray, to other epidemiologically importantwild animals, to domestic animals and human beings, also form a featureof this invention.

The new vaccine strain can be administered by different routes, i.e.which can be administered parenterally or non-parenterally, and whichappears to provide a full protection when administered before or after arabies infection. The possibility of effective oral administration withthe aim of achieving "oral vaccination" of wild animals, who are themost important vectors and/or sources of rabies, will be appreciated asan advantageous feature of the present invention.

The live virus vaccine prepared form strain 675 has the followingpossible advantages for pre-exposure vaccination:

(A) In animals:

1. Immunization of foxes and other epidemiologically important wildanimals with the aid of rabies vaccine-containing baits, said vaccinebeing enclosed in a bite-permeable container and surrounded by meatbait. 2. Oral vaccination of domestic animals, for instance stray dogs,in countries where killing them is prohibited on religious and/orlegislative grounds. 3. Large scale administration of the live virusvaccine by means of baits so that vaccination campaigns can be markedlysimplified. For instance, distribution of optionally frozen meat ballscontaining virus vaccine by aircraft. 4. Effective protection ofdomestic animals after exposure for use in countries wherein suchvaccination procedures are authorized and in countries where animals canbe kept after exposure provided they already had been immunized.

(B) In humans:

1. Fear of oral vaccination is negligible. Fear of inoculation is a veryimportant factor in several countries and causes a reduction in thenumber of patients who return for the required number of inoculations.2. Administration per os does not cause local reactions or irritationswhich form a real disadvantage for presently commercially available,prior art vaccines. 3. Easy administration on a large scale and bynon-medically trained personnel has become possible (cf. oralpoliomyelities vaccination). In this connection, the fact that onesingle administration of the vaccine has appeared to be sufficient willbe appreciated as important. For instance, the inactivated vaccines ofthe present invention may be effectively applied to humans forpre-exposure vaccination by administration of one dose of 1 ml, when thetiter of the vaccine before inactivation is about 10⁷.5 pfu/ml.Repetition of this vaccination may be necessary every year wheninfection may occur. The same does of this inactivated vaccine of thesame original titer may be administered to humans for post-exposurevaccination on the day of infection and 1,3 and 7 days thereafter forsatisfactory results.

The novel vaccines are prepared by usual procedures, i.e. in vitro cellcultures, by application of the usual and prescribed controls toeliminate bacterial and/or viral contaminations, according to the wellknown principles and international standard requirements.

The novel vaccines of the invention are preferably prepared by growingthe rabies virus in monolayers of primary or secondary chicken embryofibroblasts derived from SPF 10 day old chicken embryos. However, othercell systems which are sufficiently susceptible to rabies infection canalso be used such as other cells of avian origin or mammalian cells,e.g. the Baby Hamster Kidney 21 (21 passages) for monolayer culture orthe BHK-21 13 S (13 passages of suspension culture) for suspendedculture, but in this case only for animal vaccines. The suspended cellculture system has the great advantage of permitting production of thevirus vaccines in fermenters of large capacity which facilitatesproduction.

The vaccines can also be prepared in cells grown on micro carriers (beadcell-culture system). For example, poly dextran beads treated withmacromolecular anions prior and/or during cell growth may be used havingan adequate positive charge density for cell growth on their surface. Inthis way, a stirred suspension of cells and beads in a liquid mediumwould be inoculated with the desired virus and after propagation of thevirus, the beads are sunk to the bottom of the reactor and separatedfrom the suspension of viruses.

In a specific method for the preparation of the vaccines, the chickenembryo fibroblasts are infected with the rabies seed virus strain No.675 having a multiplicity of infection of 0.02 to 1 pfu/cell with aninfection period of at least one hour being preferred. After removal ofthe unattached virus, a maintenance medium is added, preferablyconsisting of Basal Medium Eagle (BME) in Earle's salt solution to whichappropriate amounts of antibiotics and 0.2 to 0.5% of albumin are added.Care must be taken to ensure that the pH of the maintenance medium iskept at 8.0 to 8.2 The virus-infected cells are incubated at 32°-39° C.for 3 to 10 days and the medium is then frozen at -70° C. or below todisrupt the cells and release the virus, thereby increasing the titre.The cells and cell debris are removed by filtration or centrifugationunder sterile conditions. The filtrate or supernatant liquid is storedat -70° C. and samples are taken for titration purposes by the in vitroplaquing method referred to above. It will be appreciated that othermethods may be used to disrupt the cells after incubation e.g. bysubjecting the cell suspension to ultrasonic vibration.

According to an alternative specific preparation method BHK-21 13S cellsare grown in suspension, e.g. in a spinner flask with hanging bar, usingBHK-21 medium for spinner culture to which small amounts of tryptosephosphate broth (7-15%) inactivated calf serum (7-15%) and antibiotics,such as 100 IU of penicillin and 100 micrograms of streptomycins, areadded. The cells are grown until about a level of 2×10⁶ cells/ml isreached, while the cells are agitated sufficiently to keep them insuspension and to avoid clumping together. After spinning down the cellsby a centrifuge, infection with rabies seed virus strain 675 in amultiplicity of infection between 0.01 and 1 is performed for 30 to 60minutes with constant stirring.

After infection, the cells are suspended again in the spinner flask andmaintenance medium consisting of BHK-21 spinner culture medium to whichsmall amounts of bovine serum albumin fraction V (e.g. 0.1-0.4%) andantibiotics such as kanamycin or neomycin (in an amount of e.g. 50-300micrograms) are added along with an adjustment of the pH between 7.5 and8.0. During the infection, the temperature is kept at 32° to 35° C. andpreferably at 33° C. and every day a small sample of the spinner flasksuspension is removed for examination by the immunofluorescenttechnique. Between 2 to 3 days after infection, 80 to 100% of the cellsin these samples will show a specific immunofluoresence for rabiesinclusion bodies in their cytoplasm, corresponding to a titer of 10⁸ to10⁹ pfu/ml at harvest on 4 to 6 days after infection. At harvest, theliquid is removed from the flask and stored frozen at -70° C.

A small sample is titrated after removing the cells and cell debris bythe plague titration method. If an acceptable titer was reached (between10⁷.5 and 10⁸.5 pfu/ml or higher), the cells and cell debris are removedfrom the harvest by filtration or centrifugation under sterileconditions. After addition of a suitable stabilizer, the fluid is readye.g. for distribution in vials for lyophilization of the final product.

It will be appreciated that also killed (inactivated) rabies vaccines,preferably for human use, are included within the scope of theinvention. These killed rabies vaccines may be prepared by inactivatingthe virus in the prepared culture fluids by known procedures ormodifications thereof, e.g. by addition of beta-propiolactone, formalinor acetylethyleneimine, or by ultraviolet irradiation. It will beappreciated that rabies vaccines which are derived from the virus strainof the invention may contain one or more suitable stabilizers,preservatives, buffering salts and adjuvants.

In the following examples there are described several preferredembodiments to illustrate the invention. However, it is to be understoodthat the invention is not intended to be limited to the specificembodiments.

EXAMPLE 1

Monolayers of primary or secondary SPF chicken embryo fibroblasts grownin stationary or roller bottles were infected with the rabies seed virusstrain 675 having a multiplicity of infection (m.o.i.) of 0.02 to 1pfu/cell. Lower amounts of virus could also be used but the incubationperiod was then prolonged. An infection period of at least one hour wasrequired for attachment of the seed virus to the cells. Then, theremaining unattached virus was removed and the maintenance medium wasadded, consisting of Basal medium Eagle (BME) in Earle's salt solutionto which appropriate amounts of antibiotics and 0.2% of albumin of therelevant species for which the vaccine is to be used were added, takingcare that the final mixture of the maintenance medium had a pH of 8.2.

Incubation at 33° to 35° C. for 4 to 8 days of the virus infected cellsresulted in a pronounced CPE (cytopathic effect) and the medium was thenfrozen at -70° C. Cells and cell debris were removed, after thawing ofthe product by centrifugation at 1000 g's or by filtration through amembrane filter (5 microns pore size) under sterile conditions. Thefluid, either supernatant or filtrate, was stored at -70° C. and sampleswere taken for titration purposes by the in vitro plaquing methodpreviously mentioned (Bijlenga and Joubert).

Accepted titers for vaccine batches to be used for effective vaccinationafter exposure were 10⁸ pfu/ml. For pre-exposure vaccination, minimumtiters up to 10⁷ pfu/ml were still acceptable.

The production of vaccine batches as described above was controlled bythe usual potency tests (NIH and Hable test) and for the batches to beused for post-exposure treatment an additional new potency test ofBijlenga [Symposium on Advances in Rabies Research, Atlanta, Ga., Sept.7-9, 1976, page 14] was included. In this new potency test, local streetvirus was administered intramuscularly (i.m.) to 4 week old mice,followed by a single intraperitoneal inoculation of 0.5 ml vaccinewithin 24 hours. All vaccinated mice should survive, whereasapproximately 50% of the control mice should be dead before the end ofthe 3 week testing period. Also, the usual controls for sterility testsfor possible bacterial and viral contamination of the vaccine batches,together with the procedures for the control of primary or secondarycells and cell lines employed for the production of the vaccines werefollowed according to internationally established minimum requirements.

EXAMPLE 2

BHK-21 13 S cells were grown in suspension in a Bellco spinner flaskwith hanging bar using BHK-21 medium for spinner culture to which 10%tryptose phosphate broth and 10% inactivated calf serum were added inthe presence of antibiotics (100 IU of penicillin and 100 micrograms ofstreptomycin). The cells were grown until a level of 2×10⁶ cells/ml wasreached while the cells were agitated sufficiently to keep them insuspension and to avoid clumping together.

After spinning down the cells at 800 rpm by a centrifuge, infection withrabies seed virus strain 675 in a multiplicity of infection between 0.01to 1 was performed for 45 minutes with constant stirring. Afterinfection, the cells were suspended again in the spinner flask andmaintenance medium consisting of BHK-21 spinner culture medium to which0.2% of bovine serum albumin fraction V was added together withappropriate amounts of acceptable antibiotics (i.e. kanamycin orneomycin), along with an adjustment of the pH at 7.8. During infection,the temperature was kept at 33° C. and every day a small sample of thespinner flask was removed for examination by the immunofluorescenttechnique. Between two to three days after infection, 80 to 100% of thecells in these samples showed a specific immunofluorescence for rabiesinclusion bodies in their cytoplasm, which in turn guarantees anadequate titer of 10⁸ to 10⁹ pfu/ml at harvest 4 to 6 days afterinfection. At harvest, the liquid was removed from the flask and storedfrozen at -70° C. A small sample of the harvest was kept separately fortitration after removal of the cells and cell debris by the plaquetitration method. If the titer is acceptable (between 10⁷.5 and ⁸.5pfu/ml or higher), the cells and cell debris were removed from theharvest as described previously and the fluid was ready, after adding asuitable stabilizer, for distribution into vials for lyophilization ofthe final product.

An alternative method to obtain even higher titers can be employed byinfecting either stationary complete monolayers or such monolayers inroller culture bottles. Between two and eight hours after infection, thecells were trypsinized and put into suspension culture as describedabove. Care should be taken that no cell clumping occurs. In this way,more than 2×10⁶ cells/ml can be employed which gives higher titers ofthe harvest depending upon the number of infected cells put intosuspension culture.

EXAMPLE 3

To demonstrate the use and value of the living and the inactivatedrabies vaccines of the invention, results of pre- and post-exposurevaccination experiments are listed below:

    ______________________________________                                        A. Oral vaccination of foxes with live rabies vaccine strain 675              (2ml/fox) the titer of the vaccine being 2 × 10.sup.8 pfu/ml.                       age at                                                            Identi-     time of                                                           fication    vaccin-  serum titer in IU*                                       No. of      ation    Sept.  Oct. Nov. challenge**                             foxes       Aug. 11  11     7    21   Dec. 23                                 ______________________________________                                        Orally  84      5 months 4.0  7.0  8.2  protected                             Vaccinated                                                                            86      6 months 1.3  1.6  1.4  protected                             Animals 88      6 months 2.0  2.4  3.2  protected                             controls                                                                              83      6 months                dead after                                                                    15 days                               (non-   85      6 months                dead after                            vaccinated                              17 days                               animals)                                                                              92      6 months                dead after                                                                    18 days                               ______________________________________                                         *One I.U. (International Unit) = a serum dilution of 1/300 which dilution     gives a 50% plaque reduction endpoint reading.                                **A very high dose of challenge virus (from salivary gland of naturally       infected fox). namely 1,391,610 MLD.sub.50, was used for testing the          protective value of the vaccine.                                         

This experiment shows the effectiveness of oral vaccination of youngfoxes. The immunity responses of the foxes demonstrate an adequateseroconversion and good titers. More than five months after vaccination,the three foxes were fully protected and all three control foxes diedwithin very short incubation periods which was due to the very high doseof the challenge virus inoculated intramuscularly in the right hing leg.The usual challenge dose for foxes employed was 3000 MLD₅₀.

    ______________________________________                                        B. Serological results after vaccination with inactivated strain 675          Species                                                                              Weight of             Serum titers 4 weeks                             of animal                                                                            animals in kg                                                                             dose in ml                                                                              after vaccination                                ______________________________________                                        Dog 1  6.5         2 i.m.    6.8                                              2      8           2 i.m.    6.3                                              3      12          2 i.m.    5.5                                              4      10          2 i.m.    12.3                                             5      15          2 i.m.    5.9                                              6      14          2 i.m.    4.5                                              7      17          2 i.m.    3.8                                              8      17          2 i.m.    20.5                                             Cow 1  200         5 s.c.    4.5                                              2      400         5 s.c.    8.5                                              3      300         5 s.c.    15.5                                             4      500         5 s.c.    4.8                                              5      400         5 s.c.    7.6                                              6      600         5 s.c.    8.9                                              7      500         5 s.c.    3.8                                              8      400         5 s.c.    4.7                                              ______________________________________                                    

One single injection of vaccine was administered and the indicatedtiters were expressed in I.U. as in the previous Table.

At four weeks after vaccination, the eight dogs and eight cattle allshowed a seroconversion with very high titers. The minimum titerrequired for seroconversion was established by the European Pharmacopeeat a level of 0.2 I.U./ml.

    __________________________________________________________________________    C. Protection of laboratory mice after infection with wild                    live virus (of fox origin)                                                           Interval between infection and vaccination                             Type of                                                                              1    1  2  3  4  5  6  7  8  9***                                      vaccine                                                                              hour day                                                                              days                                                                             days                                                                             days                                                                             days                                                                             days                                                                             days                                                                             days                                                                             days                                      __________________________________________________________________________    Living                                                                        vaccine                                                                       675*                                                                          Undiluted                                                                              0/10**                                                                           1/10                                                                             1/10                                                                             1/10                                                                             2/10                                                                             0/10                                                                             0/10                                                                             4/10                                                                             5/10                                                                             3/10                                      10.sup.-1                                                                            1/10                                                                   10.sup.-2                                                                            4/10                                                                   10.sup.-3                                                                            6/10                                                                   10.sup.-4                                                                            8/10                                                                   Controls                                                                             6/10 8/10                                                                             7/10                                                                             7/10                                                                             7/10                                                                             7/10                                                                             6/10                                                                             6/10                                                                             6/10                                                                             8/10                                      Inactiva-                                                                     ted                                                                           vaccine                                                                       675*                                                                          Undiluted                                                                            0/10 1/10                                                                             5/10                                                           10.sup.-1                                                                            1/10                                                                   10.sup.-2                                                                            6/10                                                                   10.sup.-3                                                                            8/10                                                                   10.sup.-4                                                                            6/10                                                                   __________________________________________________________________________     *Titer of vaccine 10.sup.8.2 pfu/ml (for inactivated vaccine the titer wa     determined before inactivation)                                               **number of deaths/number of inoculated mice.                                 ***Two vaccinations (at day 9 and 11)                                    

All mice listed in this table were inoculated intramuscularly with a foxsalivary gland rabies virus (0.1 ml of solution) which killed 60-80% ofthe controls with an incubation period of 9 to 11 days. The living andthe inactivated vaccine strain 675 were administered in one single dose(0.5 ml) intraperitoneally at various periods after the initialinfection as indicated in the Table. The living undiluted vaccineprotected still 6 days after infection which has never been observedbefore for any other commercial vaccine available. This protectionshowed that even after the rabies virus had reached the central nervoussystem, the mice can still be cured. With two vaccinations at one dayinterval and starting nine days after infection, some of the mice werestill protected.

This living vaccine could also be diluted up to ten times and affordedprotection but not longer than at 24 hours after infection. Theinactivated vaccine protected only when applied within the 24 hoursafter infection and could also be diluted but not more than ten times.

Various modifications of the vaccine and strains and processes of theinvention may be made without departing from the spirit or scope thereofand it should be understood that the invention is to be limited only asdefined in the appended claims.

I claim:
 1. A live or inactivated rabies virus vaccine wherein the virusvaccine is derived from a rabies strain novel species No. 675 depositedwith Czechoslovak National Collection of Type Culture of the Instituteof Hygiene and Epidemiology in Prague and is in an injectablepharmaceutically acceptable liquid, the amount of vaccine beingsufficient to combat rabies infections.
 2. A vaccine of claim 1 furthercontaining at least one member of the group consisting of stabilizers,preservatives, buffering salts and adjuvants.
 3. A live rabies virusvaccine for oral administration wherein the virus vaccine is derivedfrom a rabies virus strain novel species No. 675 deposited withCzechoslovak National Collection of Type Culture of the Institute ofHygiene and Epidemiology in Prague and is incorporated in baits, theamount of vaccine being sufficient to combat rabies infections.
 4. Avaccine of claim 3 wherein the bait is a meat ball.
 5. Baits containinga live rabies virus vaccine wherein the virus vaccine is derived from arabies virus strain novel species No. 675 deposited with theCzechoslovak National Collection of Type Culture of the Institute ofHygiene and Epidemiology in Prague, the amount of vaccine beingsufficient to combat rabies infections.
 6. A process for the preparationof live or inactivated rabies virus vaccines of claim 1 derived from therabies virus strain novel species no. 675 comprising preparing vaccineby growing the rabies virus in cell systems which are sufficientlysusceptible to rabies infection of cells of avian origin or mammaliancells, freezing the cell suspension at temperature of at least -70° C.and/or subjecting the cell suspension to ultrasonic vibration to disruptthe cells and release the virus, removing the cells and cell debris byfiltration or centrifugation under sterile conditions and optionallyinactivating the virus in the culture fluids.
 7. The process of claim 6wherein chicken embryo fibroblasts are infected with the rabies seedvirus strain no. 675 having a multiplicity of infection of 0.02 to 1pfu/cell, the infection period is at least one hour and adding amaintenance medium of pH 8.0 to 8.2 after removal of unattached virus,incubating the virus infected cells at 32°-39° C. for 3-10 days,freezing the cells and medium to at least -70° C. and filtering orcentrifuging under sterile conditions the mixture and optionallyinactivating the virus in the culture fluids.
 8. A process of claim 7wherein the maintenance medium is Basal medium Eagle (BME) in Earle'ssalt solution to which appropriate amounts of antibiotics and 0.2-0.5%of albumin are added.
 9. A process of claim 6 wherein Baby HamsterKidney 21 (21 passages) 13 S (13 passages of suspension culture) cells,grown in suspension in a spinner flask in a BHK-21 medium for spinnerculture to which small amounts of tryptose phosphate broth, inactivatedcalf serum and antibiotics, have been added to obtain a level of about2×10⁶ cells/ml infected with rabies seed virus strain 675 in amultiplicity of infection between 0.01 to 1 under stirring, incubatingthe virus in suspension in a maintenance medium at a pH between 7.5 and8.0 and at a temperature of 32°-35° C. for 4-6 days after infection,freezing the cells and medium at a temperature of at least -70° C. andfiltering or centrifuging the mixture under sterile conditions andoptionally inactivating the virus in the culture fluids.
 10. A method ofvaccinating warm-blooded animals against rabies comprising administeringto warm-blooded animals an antirabies effective amount of an attenuatedliquid antirabies vaccine of claim
 1. 11. The method of vaccinatingwarm-blooded animals against rabies as in claim 10 wherein the vaccineis administered orally.
 12. The method of claim 11 wherein the vaccineis incorporated in a bait.
 13. A method of protecting wild carnivoresfrom rabies infection comprising orally administering to said carnivoresan immunizing dose of the attenuated rabies vaccine strain no. 675containing a temperature stabilizer, said vaccine being enclosed in abite-permeable container and said container being surrounded by anacceptable meat bait for said carnivores.
 14. The method of claim 13wherein the wild carnivore is a fox.
 15. Method of protecting of man andanimals after exposure to rabies infections by the administration of thevaccines according to claim 1 in the form of booster shots to man andanimals, which had been vaccinated already.
 16. Method according toclaim 15, characterized in that the live rabies vaccine is appliedseveral days after exposure.